This work was supported by NSFC Grants No. 31670762 (Z.Q.).

1. Preparation of the lipid bilayer master mix
<ol>
	<li>Rinse glass vials with double-distilled water (ddH2O) and 99% ethanol and dry them in a 60 &#176;C drying oven.</li>
	<li>Make the lipid master mix by dissolving 1 g of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 100 mg of polyethylene glycol-reacted (PEGylated) lipids (18:1 of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (PEG2000 DOPE) and 25 mg of biotinylated lipids (18:1 of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (Biotinyl Cap DOPE)) into 10 mL of chloroform.</li>
	<li>Prepare 1 mL aliquots of the lipid master mix per vial and store them at -20 &#176;C in clean glass vials. 
	NOTE: Store the dissolved lipid in glass vials and cover the vial cap with parafilm during storage.</li>
</ol>
2.&#160;Preparation of liposome solution
<ol>
	<li>Rinse a 2 mL glass vial with ddH2O and 99% ethanol and dry it thoroughly in a 60 &#176;C drying oven.</li>
	<li>Rinse a 250 &#181;L glass syringe with chloroform and then transfer 200 &#181;L of the lipid master mix into the dry glass vial.</li>
	<li>Use a nitrogen spray gun to flow N2 gently while continuously rotating the vial in one direction. 
	NOTE: The N2 will help evaporate the chloroform, and the residual lipids will form a thin film on the wall of the glass vial. Adjust the nitrogen stream to be very gentle at first and increase the flow when a white film appears in the vial; complete this evaporation process within 5 min.</li>
	<li>Transfer the glass vial to a vacuum drying oven at room temperature (RT) for 16-24 h for complete removal of the chloroform from the lipids.</li>
	<li>Add 2 mL of fresh lipid buffer (10 mM Tris-HCl (pH 7.5) and 100 mM NaCl) to the glass vial and dissolve the lipids at RT for 2 h. Vortex the solution several times and transfer it to a 5 mL polypropylene culture tube.</li>
	<li>Sonicate the lipid solution on ice with a micro-tip sonicator to form small unilamellar vesicles, using the following settings: 6 s on-12 s off (20% amplitude) for 6 cycles, then 6 s on-6 s off (30% amplitude) for 3 cycles, finally 10 s on (40% amplitude). 
	NOTE: The temperature increase during sonication could result in the bursting of foam and destroy the liposomes. Therefore, check the state of the foam and the temperature and replenish the ice frequently.</li>
	<li>Filter the liposomes through a 0.22 &#181;m nylon syringe filter, aliquot, and store it at 4 &#176;C. 
	NOTE: As their mobility decreases with prolonged storage, the liposomes must be used in 2-3 months or prepared freshly before use.</li>
</ol>
3. Sequence cloning and biotinylation of Lambda DNA
<ol>
	<li>Insertion of the binding motifs into Lambda DNA
	<ol>
		<li>Digest the target fragment containing the binding motifs and Lambda DNA with NheI and XhoI. Ligate the target fragment with Lambda DNA using T4 ligase overnight at RT.</li>
		<li>Culture E.coli VCS257 cells in antibiotic-free LB medium until the OD600 reaches 0.6. Store the bacteria at 4 &#176;C for later use.</li>
		<li>Heat the ligation solution at 65 &#176;C for 20 min to denature the T4 ligase.</li>
		<li>Thaw the Lambda phage extract, mix it with the ligated product gently by pipetting with blunt tips, and incubate the mixture at RT for 2 h.</li>
		<li>Add 500 &#181;L of SM buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 8 mM MgCl2, filtered through a 0.22 &#956;m filter) and 20 &#181;L of chloroform into the packaging mixture from step 3.1.4, mix well, and centrifuge the solution. 
		NOTE: The activity of the packaging extract can be maintained at 4 &#176;C for prolonged periods.</li>
		<li>Prepare the reaction mixture by mixing 10 &#181;L of packaging extract with 90 &#181;L of SM buffer&#160;and 100 &#181;L of the VCS257 cells (from step 3.1.2) together, and incubate the mixture at 37 &#176;C for 15 min. 
		NOTE: The packaging solution concentration depends on the density of the phage plaques on the following day; it can be diluted or added as needed.</li>
		<li>Take ~5 mL of melted top agar (22 g/L NZCYM broth with 15 g/L agar) in a 15 mL tube. As soon as the top agar cools down to ~42 &#176;C, add the reaction mixture (from step 3.1.6) slowly using a blunt-end pipette and pour the liquid onto an antibiotic-free LB agar plate. Keep the plate in a 37 &#176;C incubator overnight.</li>
		<li>Confirm the positive plaques among the transparent phage plaques on the top agar plate by PCR. Transfer the positive plaques to a new top agar plate with a pipette and keep the plate in a 37 &#176;C incubator overnight.</li>
		<li>Add 400 &#181;L of VCS257 cells into 400 &#181;L of buffer containing 10 mM MgCl2 and 10 mM CaCl2, inoculate one plaque into this cell mixture, and incubate it in a 37 &#176;C shaker at 250 rpm for 15 min.</li>
		<li>Add 800 &#181;L of this suspension containing the phage plaque to 200 mL of NZCYM broth in a 2 L flask and incubate it in a 37 &#176;C shaker at 125 rpm.</li>
		<li>Measure OD600 of the bacterial suspension after ~4 h of cultivation. Keep in mind that the OD600 value will rise first and then drop to a trough at ~0.2. Stop the incubation when the OD600 value is about to rise again, add 500 &#956;L of chloroform, and shake at 80 rpm for another 5 min. 
		NOTE: The increase in OD600 after the trough will occur quickly; hence, the OD600 value must be measured more often when it decreases to 0.3 to avoid excessive VCS257 cell growth in the culture.</li>
		<li>Transfer the phage culture to a 500 mL bottle, add 11.7 g of NaCl, and shake the bottle. Incubate the bottle on ice for 10 min.</li>
		<li>Transfer the suspension equally into four 50 mL tubes. Centrifuge the tubes at 12,000 x&#160;g for 10 min. 
		NOTE: All these centrifugation steps in section 3.1 need to be done at 4 &#176;C.</li>
		<li>Transfer the supernatants to four new 50 mL tubes and centrifuge again at the same speed for 5 min.</li>
		<li>Collect the supernatants, add 10% (w/v) PEG8000 powder, and rotate at 4 &#176;C to incubate for 30 min.</li>
		<li>Centrifuge the suspensions from step 3.1.15 at 12,000 x&#160;g for 15 min to obtain the phage pellets.</li>
		<li>Resuspend the pellets in 1 mL of phage dilution buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 10 mM MgCl2) in four tubes and pour all 4 mL of these mixtures into a 50 mL tube.</li>
		<li>Add RNase A (10 mg/mL) and DNase I (4 mg/mL) to 20 &#181;g/mL and 5 &#181;g/mL final concentrations and incubate at 37 &#176;C for 30 min to degrade excess nucleic acids.</li>
		<li>Add 4.5 mL of 300 mM Tris-HCl (pH 7.5), 2.76 mL of 0.5 M ethylenediamine tetraacetic acid (EDTA), 1.67 mL of 10% sodium dodecylsulfate (SDS), and 75 &#181;L of proteinase K (10 mg/mL) into the tube, and incubate at 65 &#176;C for 10 min.</li>
		<li>Add 4.5 mL of pre-cooled 3 M potassium acetate, and incubate on ice for 10 min.</li>
		<li>Centrifuge at 8,000 x&#160;g for 10 min, and spin the supernatant for an additional 5 min at 10,000 x&#160;g.</li>
		<li>Add 70% volume of isopropanol into the collected supernatant (from step 3.1.21) with sufficient mixing, incubate at RT for 2 min, and then centrifuge at 8,000 x&#160;g for 10 min. Discard the isopropanol and spin again for 5 min. 
		NOTE: The DNA pellet will be smeared before the first 10 min centrifugation; the pellet will be concentrated at the bottom of the tube after the second 5 min centrifugation.</li>
		<li>Wash the precipitated DNA with 2-3 mL of 70% ethanol and spin down again for 1 min. Dry the pellet at 4 &#176;C for 2 h, and then resuspend the DNA in 500 &#181;L of TE buffer (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA) overnight at RT.</li>
		<li>Transfer the DNA to a 1.5 mL tube and spin it at 18,000 x&#160;g for 3 min. Transfer the DNA to a new 1.5 mL tube. Aliquot the purified Lambda DNA and store it at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Biotin-Lambda DNA preparation
	<ol>
		<li>Melt 50 &#181;L of purified Lambda DNA with cloned motifs at 65 &#176;C for 10 min. 
		NOTE: As Lambda DNA is ~48 kbp long, it must be heated before pipetting to avoid DNA breaks.</li>
		<li>Mix 50 &#181;L of Lambda DNA, 1 &#181;L of Biotin primer (5&#39; - (Phos) - AGG TCG CCG CCC - BIOTEG - 3&#39;) (100 &#181;M) and 54 &#181;L of ddH2O together. Co-incubate the mixture at 65 &#176;C for 5 min and leave it on the bench to cool down to RT.</li>
		<li>Add 12 &#181;L of 10x T4 ligase buffer and 3 &#181;L of T4 ligase, and incubate at RT for several hours or overnight.</li>
		<li>Add an equal volume of phenol-chloroform (1:1) mixture to the ligation product, mix well immediately, and centrifuge at 12,000 x&#160;g for 2 min at RT.</li>
		<li>Transfer the upper aqueous phase containing the DNA carefully to a new tube, add an equal volume of chloroform, mix well, and then centrifuge at 12,000 x&#160;g for 2 min.</li>
		<li>Transfer the upper aqueous phase to a new tube, add an equal volume of isopropanol and 10% of the volume of 3 M sodium acetate, and incubate at RT for 2 min. Centrifuge the mixture at 12,000 x&#160;g for 5 min.</li>
		<li>Remove the supernatant, wash the pellet with 75% ethanol, and then centrifuge at 12,000 x&#160;g for 5 min.</li>
		<li>Remove the 75% ethanol, air-dry the DNA at RT for 10 min, and use 120 &#181;L of TE buffer to dissolve the DNA.</li>
		<li>Add 60 &#181;L of Buffer A (30% (w/v) PEG8000 and 30 mM MgCl2) to 120 &#181;L of the solution from step 3.2.8, and rotate at 4 &#176;C for 20-24 h.</li>
		<li>Centrifuge the mixture at 18,000 g for 5 min, and remove the supernatant.</li>
		<li>Use 180 &#181;L of pre-cooled 75% ethanol to wash the pellet, and repeat step 3.2.10.</li>
		<li>Dry the pellet at RT, and use 100 &#181;L of TE150 buffer to dissolve the DNA.</li>
	</ol>
	</li>
</ol>
4. Nanofabricated zig-zag barriers
<ol>
	<li>Clean slides in acetone for 20 min by sonication and transfer them to a new glass container with 1 M NaOH for sonication for another 20 min. Mix 90 mL of H2SO4 with 30 mL of H2O2, and immerse the slides in this mixture for 30 min. Rinse the slides with acetone and isopropanol and dry the slides with N2. 
	NOTE: Mixing H2SO4 and H2O2 is an exothermic process; take adequate safety precautions during the operation.</li>
	<li>Coat the cleaned slide with polymethylmethacrylate (PMMA) 25 kDa, PMMA 49.5 kDa, and the top layer of conductive protective coating in succession. Use electron beam lithography to write the zig-zag barriers (Figure 1A).</li>
	<li>Wash the slides with ddH2O to remove the conductive protective coating and dry the slides with N2.</li>
	<li>Deposit a 30 nm layer of chromium (Cr) using a magnetron sputtering machine and remove the remaining PMMA layers with acetone. 
	&#8203;NOTE: One flowcell can be reused for more than 20 DNA Curtains experiments. To reuse the flowcell, wash it with ethanol, detergent, and NaOH.</li>
</ol>
5. Purification of EWS-FLI1 protein
NOTE: The observation of 500 nM EWS-FLI1 on Lambda DNA with 25&#215; GGAA motifs serves as a good example for the application of DNA Curtains to condensate formation. EWS-FLI1 is a fusion protein combining the N-terminal of EWSR1 (1-265) and the C-terminal of FLI1 (220-453). An mCherry tag was fused to the N-terminal of the EWS-FLI1 fusion protein for visualization.
<ol>
	<li>Clone the EWS-FLI1 gene into a reconstituted pRSF vector or any other suitable prokaryote expression vector.</li>
	<li>Transform the plasmid encoding 7x His-mCherry-EWSFLI1 into the E.coli BL21(DE3) strain; grow a single colony in 5 mL of LB medium at 37 &#176;C overnight. When the OD600 value reaches 0.6-0.8 after transferring to 2 L of LB medium, add 0.5 mM IPTG and shake the cell culture at 18 &#176;C for 16-18 h.</li>
	<li>Centrifuge the culture to remove the supernatant and resuspend the cells with lysis buffer (50 mM Tris-HCl, pH 7.5; 1 M KCl; 1 M urea; 10 mM imidazole; 1.5 mM beta-mercaptoethanol (&#946;-ME); and 5% glycerol).</li>
	<li>After sonication and centrifugation at 18000 &#215; g for ~30 min, load the supernatant onto Ni-NTA resin equilibrated in a 5-fold volume of lysis buffer, and then wash the resin with a 10-fold volume of washing buffer (50 mM Tris-HCl (pH 7.5), 1 M KCl, 1 M urea, 40 mM imidazole, 1.5 mM &#946;-ME, and 5% glycerol).</li>
	<li>Elute the bound protein with 15 mL of elution buffer (50 mM Tris-HCl (pH 7.5), 1 M KCl, 1 M urea, 500 mM imidazole, 1.5 mM &#946;-ME, and 5% glycerol) and concentrate the elution to ~500 &#181;L.</li>
	<li>Load the concentrated protein solution onto a gel filtration column and analyze the products by SDS-polyacrylamide gel electrophoresis. Quickly freeze the purified protein in liquid nitrogen and store at -80 &#176;C.</li>
</ol>
6. Preparation of a DNA Curtains flowcell
<ol>
	<li>Prepare a flowcell with zig-zag nanofabricated barriers for a DNA Curtains experiment and determine the direction of flow. To do this, connect the input and output tubes in the correct direction.</li>
	<li>Wash the flowcell with 2.5 mL of lipid buffer using two 3 mL syringes. Ensure there is no bubble in the flowcell.</li>
	<li>Remove the syringe from the output, and inject 1 mL of the liposome solution (40 &#181;L of liposome stock from section 2 and 960 &#181;L of lipid buffer) three times with 5 min incubation after each injection.</li>
	<li>For the healing step, wash the flowcell with 2.5 mL of lipid buffer slowly, and incubate at RT for 30 min. 
	NOTE: The healing time can be longer if necessary; if any bubbles appear, perform double-healing by repeating steps 5.3 and 5.4 to remove the bubbles.</li>
	<li>Prepare 30 mL of bovine serum albumin (BSA) buffer (40 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM dithiothreitol, and 0.5 mg/mL BSA for surface passivation). After 30 min of healing, wash the flowcell with 2.5 mL of BSA buffer from the output side. 
	NOTE: The BSA stock is a 20 mg/mL solution stored at 4 &#176;C and must be used within 1-2 weeks. The BSA concentration in the buffer can be adjusted according to the surface condition.</li>
	<li>Inject 800 &#181;L of streptavidin buffer (10 &#181;L of a 1 mg/mL streptavidin stock, 790 &#181;L of BSA buffer) into the flowcell from the input side in two steps with 10 min of incubation after each step. 
	NOTE: The streptavidin anchors the DNA onto the lipids due to its multivalency by bridging between the biotinylated lipids and the biotinylated DNA.</li>
	<li>Wash the flowcell with 2.5 mL of BSA buffer to remove all free streptavidin molecules.</li>
	<li>Dilute the Biotin-Lambda DNA with cloned motifs from section 3 using BSA buffer (2.5 &#181;L of DNA and 998 &#181;L of BSA buffer). Inject Lambda DNA 4 times slowly at 5 min intervals.</li>
	<li>Turn on the microscope and the scientific Complementary Metal Oxide Semiconductor (sCMOS) system during the injection. Wash the tubing with 10 mL of ddH2O. Rinse the prism and the tubing connector with water, 2% liquid cuvette cleaner, and 99% ethanol. Prepare the imaging buffer by adding KCl and double-stranded DNA dye into the BSA buffer to obtain final concentrations of 150 mM and 0.5 nM, respectively, and take up at least 20 mL of the imaging buffer into a 30 mL syringe.</li>
	<li>Set up the flowcell on the microscope stage and connect it to the microfluidic system.</li>
	<li>Use a flow rate of 0.03 mL/min to flush the DNA molecules to the barrier for 10 min, incubate for 30 min with the flow stopped, and then switch it off to let the DNA diffuse laterally. 
	&#8203;NOTE: The purpose of this 30 min incubation is to let the DNA molecule distribute more evenly in front of the barrier through simple diffusion because DNA molecules tend to accumulate on the side of the barrier that is closed to the input where they are flushed in. The incubation time can be adjusted as necessary.</li>
</ol>
7. Imaging of EWS-FLI1 condensation formation on DNA Curtains
<ol>
	<li>Open the imaging software, find and mark the positions of the 3 &#215; 3 zig-zag patterns under bright-field.</li>
	<li>Turn on the flow at 0.2 mL/min to stain the DNA with the double-stranded DNA dye for 10 min.</li>
	<li>Dilute mCherry-EWS-FLI1 with the imaging buffer to the concentration of 100 nM in 100 &#181;L in the tube.</li>
	<li>Load the protein sample through the valve with a 100 &#181;L glass syringe, and change the flow rate to 0.4 mL/min.</li>
	<li>Turn on the 488 nm laser and pre-scan each region to check the DNA distribution state; select the region in which the DNA molecules distribute evenly. Set the laser power to 10% for the 488 nm laser and 20% for the 561 nm laser. Use the power meter to measure the real laser power near the prism: 4.5 mW for the 488 nm laser and 16.0 mW for the 561 nm laser.</li>
	<li>Image acquisition
	<ol>
		<li>Start acquiring images at 2 s intervals with both 488 nm and 561 nm lasers simultaneously.</li>
		<li>Change the valve from manual mode to injection mode to let the imaging buffer flush the protein sample into the flowcell after 60 s. 
		&#8203;NOTE: This process will take ~30 s, and the field-of-view will be covered with mCherry signals as soon as the EWS-FLI1 proteins reach the flowcell.</li>
		<li>To remove free EWS-FLI1, keep washing the flowcell with the imaging buffer for 5 min with only the 561 nm laser switched on. Stop the flow and incubate at 37 &#176;C for 10 min.</li>
		<li>Turn on the flow at 0.4 mL/min to let the DNA extend, and acquire images at 2 s intervals between different frames to obtain high-throughput data of EWS-FLI1 condensate formation.</li>
	</ol>
	</li>
</ol>
8. Intensity analysis for mCherry-EWS-FLI1
<ol>
	<li>Import the data as image sequences into ImageJ software, pick out the puncta at 25&#215; GGAA sites in an 8&#215; 8 pixels square, and save the image in .tif format.</li>
	<li>Calculate the intensity as the summation of the intensity in the 8&#215;8 pixels square, including the whole puncta, and remove the background by subtracting the intensity of background in an area of the same size near the 8 x 8 pixels square.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+recombination+intermediates+with+single-stranded+DNA+curtains.">Visualizing recombination intermediates with single-stranded DNA curtains.</a> Methods. 105, 62-74 (2016).</li><li>Ma, C. J., Gibb, B., Kwon, Y., Sung, P., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+dynamics+of+human+RPA+and+RAD51+on+ssDNA+during+assembly+and+disassembly+of+the+RAD51+filament.">Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament.</a> Nucleic Acids Research. 45 (2), 749-761 (2017).</li><li>Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+interrogation+by+the+CRISPR+RNA-guided+endonuclease+Cas9.">DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.</a> Nature. 507 (7490), 62-67 (2014).</li><li>Larson, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+droplet+formation+by+HP1+alpha+suggests+a+role+for+phase+separation+in+heterochromatin.">Liquid droplet formation by HP1 alpha suggests a role for phase separation in heterochromatin.</a> Nature. 547, 236-240 (2017).</li><li>Zuo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loci-specific+phase+separation+of+FET+fusion+oncoproteins+promotes+gene+transcription.">Loci-specific phase separation of FET fusion oncoproteins promotes gene transcription.</a> Nature Communications. 12 (1), 1491 (2021).</li><li>Collins, B. E., Ye, L. F., Duzdevich, D., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+curtains:+novel+tools+for+imaging+protein-nucleic+acid+interactions+at+the+single-molecule+level.">DNA curtains: novel tools for imaging protein-nucleic acid interactions at the single-molecule level.</a> Methods in Cell Biology. 123, 217-234 (2014).</li><li>Greene, E. C., Wind, S., Fazio, T., Gorman, J., Visnapuu, M. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+curtains+for+high-throughput+single-molecule+optical+imaging.">DNA curtains for high-throughput single-molecule optical imaging.</a> Methods in Enzymology. 472, 293-315 (2010).</li><li>Gangwal, K., Close, D., Enriquez, C. A., Hill, C. P., Lessnick, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergent+properties+of+EWS/FLI+regulation+via+GGAA+microsatellites+in+Ewing's+sarcoma.">Emergent properties of EWS/FLI regulation via GGAA microsatellites in Ewing's sarcoma.</a> Genes &amp; Cancer. 1 (2), 177-187 (2010).</li><li>Sizemore, G. M., Pitarresi, J. R., Balakrishnan, S., Ostrowski, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ETS+family+of+oncogenic+transcription+factors+in+solid+tumours.">The ETS family of oncogenic transcription factors in solid tumours.</a> Nature Reviews. Cancer. 17 (6), 337-351 (2017).</li><li>Roy, R., Hohng, S., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-molecule+FRET.">A practical guide to single-molecule FRET.</a> Nature Methods. 5 (6), 507-516 (2008).</li><li>Huang, B., Bates, M., Zhuang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+fluorescence+microscopy.">Super-resolution fluorescence microscopy.</a> Annual Review of Biochemistry. 78, 993-1016 (2009).</li><li>Bustamante, C., Bryant, Z., Smith, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+years+of+tension:+single-molecule+DNA+mechanics.">Ten years of tension: single-molecule DNA mechanics.</a> Nature. 421 (6921), 423-427 (2003).</li><li>Bustamante, C., Chemla, Y. R., Moffitt, J. R., Selvin, P. R., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+dual-trap+optical+tweezers+with+differential+detection.">High-resolution dual-trap optical tweezers with differential detection.</a> Single-Molecule Techniques: A Laboratory Manual. , (2008).</li><li>Zhao, Y. L., Jiang, Y. Z., Qi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+biological+reaction+intermediates+with+DNA+curtains.">Visualizing biological reaction intermediates with DNA curtains.</a> Journal of Physics D: Applied Physics. 50 (15), 153001 (2017).</li></ol>

The authors have no conflicts of interest.

As single-molecule approaches are extremely sensitive to the contents of the reaction system, extra effort must be invested to ensure good quality of all the materials and solutions during the DNA Curtains experiments, especially the lipids prepared in sections 1 and 2 and the buffers used in section 5. Reagents of higher purity must be used to prepare buffers, and buffers must be freshly prepared for the single-molecule assay
When 500 nM mCherry-labeled EWS-FLI1 was flushed into the chamber, ...

Transcriptional regulation is a crucial step for precise gene expression in living cells. Many factors, such as chromosomal modification, transcription factors (TFs), and non-coding RNAs, participate in this complicated process1,2,3. Among these factors, TFs contribute to the specificity of transcriptional regulation by recognizing and binding to specific DNA sequences known as promoters or enhancers and subsequently recruiting other functional proteins to activate or repress transcription4,5,6,7. How these TFs manage to search for their target sites in the human genome and interact with DNA coated with histones and non-histone DNA-binding proteins has perplexed scientists for decades. In the past few years, several classical models for the target search mechanism of TFs have been built to describe how they &#34;slide,&#34; &#34;hop,&#34; &#34;jump,&#34; or &#34;intersegment transfer&#34; along the DNA chain8,9,10,11. These models are focused on the searching behavior on the DNA of one single TF molecule. However, recent studies show that some TFs undergo liquid-liquid phase separation (LLPS) either alone in the nucleus or with the Mediator complex12. The observed droplets of TFs are associated with the promoter or enhancer regions, highlighting the role of biomolecular condensate formation in transcription and the three-dimensional genome13,14,15. These biomolecular condensates are linked to membrane-lacking compartments in vivo and in vitro. They are formed via LLPS, in which modular biomacromolecules and intrinsically disordered regions (IDRs) of proteins are two main driving forces of multivalent interactions16. Thus, TFs not only search DNA but also function synergistically within these condensates4,17,18. To date, the biophysical property of these transcription condensates on DNA remains unclear.
Therefore, this study aimed to apply a single-molecule method-DNA Curtains-to directly image the formation and dynamics of the transcription condensates formed by TFs on DNA in vitro. DNA Curtains, a high-throughput in vitro imaging platform to study the interaction between proteins and DNA, has been applied in DNA repair19,20,21, target search22, and LLPS17,23,24. The flowcell of DNA Curtains is coated with biotinylated lipid bilayers to passivate the surface and allow the biomolecules to diffuse on the surface. The nanofabricated zig-zag patterns limit the movement of DNA. Biotinylated Lambda DNA substrates can align along the barrier edges and be stretched by the oriented buffer flow. The same starting and ending sequences of all the molecules allow the tracking of the protein on DNA and describe the position distribution of the binding events25,26. Moreover, the combination of DNA Curtains with total internal reflection fluorescence microscopy (TIRFM) helps minimize the background noise and detect signals at a single-molecule level. Thus, DNA Curtains could be a promising method to investigate the dynamics of transcription condensate formation on DNA motifs. This paper describes the example of an FUS/EWS/TAF15 (FET) family fusion oncoprotein, EWS-FLI1, generated by chromosomal translocation. Lambda DNA containing 25&#215; GGAA-the binding sequence of EWS-FLI127- was used as the DNA substrate in the DNA Curtains experiments to observe how EWS-FLI1 molecules undergo LLPS on DNA. This manuscript discusses the experimental protocol and data analysis methods in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>488 nm diodepumped solid-state laser</td>
			<td>Coherent</td>
			<td>OBIS488LS</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm diodepumped solid-state laser</td>
			<td>Coherent</td>
			<td>OBIS561LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Rhawn</td>
			<td>R003215-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated DOPE</td>
			<td>Avanti</td>
			<td>870273P</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Amresco</td>
			<td>1595C027</td>
			<td></td>
		</tr>
		<tr>
			<td>Coating Electra 92</td>
			<td>Allresist GmbH</td>
			<td>AR-PC 5090.02</td>
			<td>The conductive protective coating</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I bovine</td>
			<td>Sigma</td>
			<td>D5139-2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti</td>
			<td>850375P</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hellmanex III</td>
			<td>Sigma</td>
			<td>Z805939-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>60130</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda DNA</td>
			<td>NEB</td>
			<td>N3013S</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda Packing Extracts</td>
			<td>Epicentre</td>
			<td>MP5120</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>s3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoport</td>
			<td>Idex</td>
			<td>N-333-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NheI-HF</td>
			<td>NEB</td>
			<td>R3131S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Inverted Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>NZCYM Broth</td>
			<td>Sigma</td>
			<td>N3643-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-2000 DOPE</td>
			<td>Avanti</td>
			<td>880130P-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>Amresco</td>
			<td>25322-68-3</td>
			<td></td>
		</tr>
		<tr>
			<td>PMMA 200K, ETHYL LACTATE 4%</td>
			<td>Allresist GmbH</td>
			<td>AR-P 649.04</td>
			<td></td>
		</tr>
		<tr>
			<td>PMMA 950K, ANISOLE 2%</td>
			<td>Allresist GmbH</td>
			<td>AR-P 672.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Prime 95B Scientific CMOS camera</td>
			<td>PHOTOMETRICS</td>
			<td>Prime95B</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase K</td>
			<td>NEB</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica glass slide</td>
			<td>G.Finkenbeiner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Six-way injection valve</td>
			<td>Idex</td>
			<td>MXP9900-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo</td>
			<td>S888</td>
			<td>Diluted with ddH2O</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>Pump11 Elite</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>NEB</td>
			<td>R0146V</td>
			<td></td>
		</tr>
		<tr>
			<td>YOYO-1 Iodide (491/509)</td>
			<td>Invitrogen</td>
			<td>Y3601</td>
			<td>Diluted with DMSO</td>
		</tr>
	</tbody>
</table>

single-molecule imaging of ews-fli1 condensates assembling on dna

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

The schematic of DNA Curtains is shown in Figure 1A, Figure 1B, and Figure 1D. The cloned target sequence containing 25 uninterrupted repeats of GGAA is found in the NORB1 promoter in Ewing sarcoma. This target sequence is crucial for EWS-FLI1 recruitment28. EWS-FLI1 molecules were visualized by detecting the mCherry-labeled EWS-FLI1 signals obtained with a 561 nm laser (Figure 1...

Watch this Scientific Journal Video about Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA at JoVE.com

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

single-molecule-imaging-of-ews-fli1-condensates-assembling-on-dna

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Biochemistry

2.3K Views.  Peking University. Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

Video: Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Imaging the Motion of Fully Reconstituted Cdc45/Mcm2-7/GINS (CMG)

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication ...

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper ...

In Situ Nucleosome Reconstitution for Single-Molecule Studies

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...